Int. J. Mol. Sci. 2015, 16, 5572-5589; doi:10.3390/ijms16035572 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article Regulatory Effects of Caffeic Acid Phenethyl Ester on Neuroinflammation in Microglial Cells Cheng-Fang Tsai 1,†, Yueh-Hsiung Kuo 1,2,†, Wei-Lan Yeh 3, Caren Yu-Ju Wu 4, Hsiao-Yun Lin 5, 4 4 4 1 5,6, Sheng-Wei Lai , Yu-Shu Liu , Ling-Hsuan Wu , Jheng-Kun Lu and Dah-Yuu Lu * 1 Department of Biotechnology, Asia University, Taichung 413, Taiwan; E-Mails: [email protected] (C.-F.T.); [email protected] (Y.-H.K.); [email protected] (J.-K.L.) 2 Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan 3 Department of Cell and Tissue Engineering, Changhua Christian Hospital, Changhua 500, Taiwan; E-Mail: [email protected] 4 Graduate Institute of Basic Medical Science, College of Medicine, China Medical University, Taichung 404, Taiwan; E-Mails: [email protected] (C.Y.-J.W.); [email protected] (S.-W.L.); [email protected] (Y.-S.L.); [email protected] (L.-H.W.) 5 Graduate Institute of Neural and Cognitive Sciences, China Medical University, Taichung 404, Taiwan; E-Mail: [email protected] 6 Department of Photonics and Communication Engineering, Asia University, Taichung 413, Taiwan † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +886-4-2205-3366 (ext. 8206); Fax: +886-4-2207-1507. Academic Editor: Guido R. M. M. Haenen Received: 12 December 2014 / Accepted: 28 February 2015 /Published: 11 March 2015 Abstract: Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effects on microglia are therefore considered as a potential strategy to counter balance neurodegenerative progression. Caffeic acid phenethyl ester (CAPE), a natural phenol in honeybee propolis, is known to possess antioxidant, anti-inflammatory and anti-microbial properties. Accordingly, the current study intended to probe the effects Int. J. Mol. Sci. 2015, 16 5573 of CAPE on microglia activation by using in vitro and in vivo models. Western blot and Griess reaction assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (NOS), cyclooxygenase (COX)-2 and the production of nitric oxide (NO). Administration of CAPE resulted in increased expressions of hemeoxygenase (HO)-1and erythropoietin (EPO) in microglia. The phosphorylated adenosine monophosphate-activated protein kinase (AMPK)-α was further found to regulate the anti-inflammatory effects of caffeic acid. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. The current study has evidenced several possible molecular determinants, AMPKα, EPO, and HO-1, in mediating anti-neuroinflammatory responses in microglial cells. Keywords: microglia; neuroinflammation; neurodegeneration; caffeicacid 1. Introduction Microglia, as sentinel cells in the brain, react to pathogens or diffusible mediators regulated by neighboring cells such as astrocytes or neurons [1,2]. Rapid microglial activation and associated inflammatory reactions are responses to combat the effect of insults, and contribute to immune-defense and tissue repair in the central nervous system (CNS) [3]. This acute activation is therefore considered to be protective [4]. By contrast, persistent microglial activation will ultimately results in vast production of proinflammatory mediators, chemokines and recruitment of peripheral immune cells [5] to the brain that characterizes chronic neurodegenerative diseases including multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease [6–8]. Manifestations of activated microglia lies in its phenotypical alternation from a resting ramified state to active form marked by increased neurotoxic factors including the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 [9,10]. Increasing evidence has revealed that iNOS mediates NO production [11], and induction of COX-2 in activated microglia causes the progressive neuronal damage [12] and brain tumor [13]. Therefore, pharmaceuticals that can deliver inhibitory effects on microglia are considered as a potential strategy to counter balance neurodegenerative progression [14]. Heme oxygenase (HO)-1, a stress-inducible protein, catalyzes a rate-limiting step of cellular oxidative agents against inflammatory responses or oxidative challenges [15]. Regulatory effects on inflammation and neuroprotection of HO-1 were also addressed in our previous study using microglia [16–18] and neuronal cells [19,20]. Other anti-inflammatory advances have shown signature neuroprotection of HO-1 via negative regulation of iNOS expression and iNOS-dependent NO production [21,22]. Moreover, induction of HO-1 has demonstrated protection from hypoxia-induced pulmonary hypertension by effectively modifying macrophage activation [23]. Erythropoietin (EPO) was originally recognized for its central role in erythropoiesis, which is crucial for the development of erythroid progenitors [24]. EPO has been identified as a neurotrophic factor and prevents neuronal apoptosis [25] by its ability to suppress tight junction openings and endothelial cell swelling [26]. Similarly beneficially, EPO was shown to ameliorate ischemic-induced microglial activation through TNF-α and IL-6 inhibition by up-regulation of HO-1 [27–29]. EPO maintains microglial integrity during oxygen-glucose deprivation [30]. The Int. J. Mol. Sci. 2015, 16 5574 AMP-activated protein kinase (AMPK) in the brain and peripheral nervous system is an energy sensor that responds to ATP-depleting processes such as cellular stress or increases in the AMP/ATP ratio [31]. Accumulating evidence demonstrates that AMPK regulates cell migration, synaptic plasticity, and inflammatory responses [32]. Importantly, AMPKα is crucial for phagocytosis-induced macrophages from a pro-inflammatory to anti-inflammatory phenotype at the time of modulation of the inflammatory response [33]. The anti-inflammation of AMPKα on microglia has been addressed in our previous studies to be either activated by berberine or lycopene [16,34]. Induction of endogenous protective molecules in microglia may be useful as a therapeutic strategy in neuroinflammation and has benefits in neuroprotection. Caffeic acid phenethyl ester (CAPE), as one of major bioactive ingredients of propolis, possesses unique biological activities including anti-oxidant, anti-inflammation, anti-tumor, and immune regulation [35–37]. As a natural food constituent, CAPE displays functional and structural similarity to polyphenol demonstrated to effectively activate AMPKα phosphorylation [38] that is beneficial over metabolic diseases, and cardiovascular diseases [39,40]. CAPE exerts strong anti-oxidant effects through blocking reactive oxygen species [41,42]. The anti-inflammatory activity of CAPE has been shown to inhibit arachidonic acid mediated COX-2 production [43], and NF-κB-dependent transcription. CAPE and its derivatives also have been reported to exert anti-oxidative and anti-inflammation properties in many cells [44,45]. Furthermore, several studies have suggested that CAPE holds a potential in neurodegenerative diseases [46]. It has been demonstrated that CAPE protects the integrity of the blood-brain barrier and protects against the loss of dopaminergic neurons in animal models [47,48]. In addition, CAPE was shown to exert direct neuronal protection through up-regulation of endogenous antioxidant and growth factor expression [35]. Our previous studies have addressed the effects of CAPE derivatives modulating inflammatory homeostasis [17]. Although the beneficial effects of CAPE in the nervous system have been investigated, detailed mechanisms of the regulation of microglia activation is not yet clear. In the present study, we addressed whether, in addition to inhibiting cytokine production, endogenous protective molecules also contribute to CAPE-regulated anti-inflammatory responses in microglial cells. Findings from the current study reveal that CAPE activates AMPKα phosphorylation concurrently with the up-regulation of EPO and HO-1 that may contribute to the microglial anti-inflammatory response. 2. Results 2.1. Caffeic Acid Phenethyl Ester (CAPE) Attenuates Microglial Activation Dose Dependently without Obvious Cytotoxicity Prior to study the effect of CAPE on microglia, the potential toxicity of CAPE was examined. Microglial cells were pretreated with CAPE (0.1 to 1.75 μM) for 30 min followed by lipopolysaccharide (LPS) treatment for another 24 h. The results showed that cell viability was not different from that of control cells following CAPE treatment (0.1 to 1.75 μM) as shown in Figure 1A. In addition, CAPE decreased nitric oxide production dose dependently after 24 h LPS stimulation (Figure 1B). To determine the effect of CAPE on proinflammatory mediator production, protein expressions of iNOS (Figure 1C) and COX-2 (Figure 1D) denoted microglial activation were examined, and found to display stepwise Int. J. Mol. Sci. 2015, 16 5575 decrement in accordance with CAPE concentrations. To further evaluate the impact of CAPE on cytokine production following LPS stimulation, cells were treated with LPS in the presence or absence of CAPE,